Biological processes offer multiple advantages compared with conventional chemistry [1
]. Proper and well-designed enzyme immobilization processes can improve the characteristics, capacity, and performance of biocatalysts in aspects such as reuse, stability, prevention of product contamination with enzyme, minimization of allergenicity, generation of new catalytic properties, increased specificity and selectivity, among others [2
]. Immobilization strategies include molecular cross-linking, encapsulation, entrapment, and binding to solid supports by physical adsorption or formation of covalent linkages [2
Commercial epoxy-activated acrylic resins such as Eupergit®
C and Eupergit®
C 250 L have excellent properties for covalent immobilization of proteins, and in some cases provide good stabilization of the biomolecules. The above-mentioned supports are formed by spherical macroporous particles possessing large internal surfaces and characterized by a low water uptake. They differ in their internal morphology and epoxy group content, which determine important differences in their properties. Eupergit C 250 L has larger pores and a lower number of oxirane groups than Eupergit C [6
Protein immobilization onto epoxy-activated acrylic supports occurs as a two-step mechanism. The primary event is rapid physical adsorption of the protein onto the resin; in a second step, a covalent reaction takes place between the adsorbed protein and the support [8
]. Epoxy groups react with amino groups in proteins under very mild experimental conditions (e.g., pH 7.5), with minimal chemical modification of the protein and formation of very stable secondary amine bonds [6
]. Multipoint covalent attachment followed by blocking the remaining epoxide moieties with hydrophilic reagents can generate even more stable enzyme derivatives under controlled conditions [3
]. The geometric characteristics of epoxy activated resins also favor multisubunit immobilization [3
Many efforts have been focused on the development and application of covalent immobilization technologies for the biotransformation of lactose, the main carbohydrate of milk, whey, and dairy products. Lactose is a disaccharide of very low solubility and low sweetness compared with its hydrolysis products, d
-glucose and d
-galactose. In the food industry, lactose hydrolysis increases solubility and sweetness, resulting in improved sensory characteristics of foods containing hydrolyzed lactose from milk or whey. A major consideration is that more than 70% of human adults worldwide have intestinal dysfunction when they consume milk [10
The enzymatic hydrolysis of lactose by β-galactosidases (EC 18.104.22.168) takes place in mild conditions and does not cause undesired flavors, odors, or colors. However, enzyme inhibition by the reaction products remains one of the main disadvantages of reactions catalyzed by β-galactosidases. Current research into the processing of dairy products is focused on the biotransformation of lactose into products of interest other than glucose and galactose [11
Commercial preparation of β-galactosidase from Bacillus circulans
(Biolacta N-5, Daiwa Kasei, Osaka, Japan) consists of various isoforms, but β-galactosidase-I is the main form in terms of abundance [12
]. The activity of this isozyme is not affected by an ionic environment (an advantage compared with certain Kluyveromyces
β-galactosidases that are inhibited by divalent calcium); its optimal reaction conditions are 44 °C and pH 5.5–6.5 [13
]. The enzyme also catalyzes the regioselective synthesis of oligosaccharides by formation of β (1–4) linkages [14
]. Recently, the β-galactosidase from Bacillus circulans
was covalently immobilized onto a commercially available matrix, Eupergit C 250 L, with high protein immobilization yields (90%–99%) and high activity immobilization yields (around 80%–90%) [16
-Arabinose isomerase (EC 22.214.171.124) is an intracellular enzyme that catalyzes the partial isomerization of l
-arabinose to l
-ribulose in vivo [17
]. In vitro experiments have shown that this biocatalyst can also convert d
-galactose to d
-tagatose, a rare sugar with promising nutraceutical properties [23
-Tagatose is a ketohexose of great interest for both technological and nutritional reasons, and many alternative processes for obtaining it, apart from chemical synthesis [25
], have been studied in recent years [23
]. Biological conversion of d
-galactose to d-
tagatose employing the enzyme l
-arabinose isomerase is the most economically viable biological d
-tagatose manufacturing process so far. However, biological production of d
-tagatose requires the development of optimized biotransformation processes with stabilized biocatalysts [26
]. It has recently been reported that l
-arabinose isomerase from Enterococcus faecium,
a bacterial enzyme with interesting properties, was produced and purified by affinity techniques [28
-glucose) isomerase (d
-xylose keto isomerase, EC 126.96.36.199) is an intracellular enzyme that catalyzes the partial and reversible in vivo isomerization of d
-xylose into d
-xylulose and in vitro of d
-glucose into d
], and therefore has the potential to transform glucose, the other product (along with galactose) of enzymatic lactose hydrolysis.
Certain authors have explored the potential of the combined use of these biocatalysts; e.g., Rhimi et al. (2007) reported the co-expression in E. coli
-arabinose isomerase from Bacillus stearothermophilus
US100 and a mutant d
-xylose isomerase from Streptomyces
SK followed by cell immobilization on alginate beads [22
]; and Xu et al. (2016) developed the co-expression of the β-galactosidase from Thermus thermophilus
HB27 and the l
-arabinose isomerase from Lactobacillus fermentum
CGMCC 2921 [27
The efficient immobilization or co-immobilization of enzymes that participate in cascade reactions can mimic or conveniently modify in vitro processes from a technological point of view [30
]. However, the choice of a particular strategy strongly depends on each case, even for one pot configuration. While co-immobilization may be preferred because of the local increase in concentration of secondary substrates and derived kinetic advantages, the intrinsic complexity of the system—including differences in multiple enzyme stability, requirements for homogeneous immobilization protocols, and reduction in stability due to undesired interactions—creates great difficulties for some applications [31
In the present paper, we report the development of a tri-enzymatic system composed of β-galactosidase, d
-glucose) isomerase, and l
-galactose) isomerase, immobilized individually onto oxirane-carrying Eupergit resins [6
]. The resulting complex biocatalysts were characterized. Functional characteristics were determined and compared to those for soluble enzymes for each of the enzymes separately immobilized by covalent attachment to Eupergit C or Eupergit C 250 L. Subsequently, the capacity of the single-immobilizates to produce d
-tagatose and d
-fructose from whey lactose under sequential or mono-reactor operative conditions was studied.
3. Materials and Methods
The enzyme β-galactosidase from B. circulans (Biolacta N-5) was kindly provided by Daiwa Kasei (Osaka, Japan), d-xylose (d-glucose) isomerase was purchased from Hampton Research (Aliso Viejo, CA, USA). Eupergit C and Eupergit C 250 L were a gift from Röhm Pharma (Darmstadt, Germany). O-Nitrophenyl-β-d-galactopyranoside (ONPG), α-lactose, d-galactose, d-tagatose, d-glucose, d-fructose, l-cysteine hydrochloride, and carbazole were purchased from Sigma (St. Louis, MO, USA); bicinchoninic acid (BCA reagent) was from Pierce (Rockford, IL, USA); and all other chemicals were of analytical or HPLC grade. The enzymatic kit for glucose determination was from Spinreact S.A. (Girona, Spain). Cheese wheys were kindly supplied by CONAPROLE (Cooperativa Nacional de Productores de Leche, Uruguay).
3.2. Sugar Analysis
The glucose and the total ketohexose contents in the samples were measured by Trinder’s assay and the cysteine-carbazole-sulfuric acid method, respectively [45
Analysis and characterization of sugars by HPLC was performed in a Waters-Millipore apparatus (Waters-Millipore Corp., Burlington, NC, USA) equipped with a carbohydrate analysis column (Rezex RCM, Phenomenex, Torrance, CA, USA) and a RI-detector, at 80 °C and a flow rate of 0.6 mL/min, employing distilled water as mobile phase and lactose, d-glucose, d-galactose, d-tagatose, and d-fructose as standards (Sigma Chem. Co., St. Louis, MO, USA).
3.3. Protein Determination
The protein concentration in soluble samples and gel derivatives were determined using the BCA reagent [47
3.4. Soluble Enzyme Treatment
The commercial β-galactosidase was partially purified as indicated by Torres and Batista-Viera [16
]. The crude extract of l
-arabinose isomerase from E. faecium
was obtained as described in Manzo et al. [28
] and purified by affinity chromatography on l
]. The commercial enzyme preparation of d
-xylose isomerase was diluted 10-fold in 0.1 M potassium phosphate buffer pH 7.5, and gel filtered on Sephadex G-25 to remove ammonium sulphate and low molecular weight additives.
3.5. Enzyme Immobilization
Immobilization of each one of the three enzymes onto Eupergit C or Eupergit C 250 L was carried out at 25 °C with shaking in 1 M potassium phosphate buffer pH 7.5 for 24 h. Applied loads were 30 mg/g of gel (β-galactosidase), 4 mg/g of gel (l
-arabinose isomerase), and 2 mg/g of gel (d
-xylose isomerase). Derivatives were collected on a sintered-glass filter and washed with 1 M potassium phosphate buffer pH 7.5 and 0.1 M sodium phosphate buffer pH 7.5. The enzyme derivatives were stabilized by alkaline treatment and blocking with glycine as indicated in a previous report [16
“Protein Immobilization Yield” was defined as the ratio (as percentage) of the amount of protein found on the gel to the amount of protein applied. “Immobilized Activity Yield” was defined as the ratio (as percentage) of the activity expressed by the gel derivative to the amount of total applied activity. Thus, “Protein Immobilization Yield” and “Immobilized Activity Yield” for the three enzymes were calculated as [Pgel
] × 100 and [Agel
] × 100, where P corresponds to protein amount and A to enzyme activity [16
3.6. Enzyme Assays
β-Galactosidase and lactase activities as well as l
-galactose) isomerase activity for soluble or immobilized enzymes were assayed as previously reported [16
-Glucose isomerase activity was determined by measuring the amount of d
-fructose generated from d
-glucose. The reaction mixture contained 5 mM MgCl2
, 1 mM CoCl2
, 500 mM d
-glucose, 200 µL of appropriately diluted enzyme preparation, and 50 mM sodium phosphate buffer pH 7.5 to bring the final volume to 1 mL. The assay was done by incubating the reaction mixture at 25 °C for 1 h [42
]. The enzymatic reaction was stopped (in the case of soluble enzyme) by boiling the samples for 3 min. The amount of d
-fructose produced was determined spectrophotometrically at 560 nm by the cysteine-carbazole-sulfuric acid method [46
]. The immobilized enzyme activity was assayed by the same method. One unit of glucose isomerase activity was defined as the amount of enzyme catalyzing the formation of 1 µmol of keto-sugar per minute under the specified conditions.
3.7. Properties of Insoluble Derivatives
The influence of pH and temperature on the activity and stability of native enzymes and immobilized derivatives was studied.
3.7.1. Effect of Temperature on Activity
The influence of temperature on the activity of soluble enzymes and immobilized derivatives was determined in the range of 20–60 °C under the pH conditions applicable for each assay. For assays, amounts of 20–50 mg of insoluble filter dried biocatalyst were used.
3.7.2. Thermal Stability
The thermostability assays were performed at different temperatures in the range 20–60 °C in activity buffer, by incubating soluble enzymes or immobilized derivatives in a shaking bath. The inactivation kinetics of biocatalysts were evaluated for 1 to 55 h, and the corresponding half-lives calculated, according to the Sadana-Henley model [50
], as described in a previous report [16
3.7.3. Optimum pH and pH Stability
The influence of pH on the activity of native enzymes and insoluble derivatives was monitored in the pH range 4–9 at 25 °C, using the corresponding substrate, according to previous reports [16
]. The effect of pH on stability was determined in the same pH range by measuring the residual activity after incubating free or immobilized catalyst at the selected pH for 1–24 h at 25 °C. For pH activity or stability assays of insoluble derivatives, amounts of 20–50 mg of filter dried biocatalyst were used. In addition, the effect of pH was evaluated under operative conditions at 50–60 °C in the range 4.0–9.0.
3.7.4. Determination of Kinetic Parameters
The kinetic parameters were determined using the corresponding substrate (0–500 mM) in 0.1 M phosphate buffer pH 7.0 at temperatures in the range of 30–50 °C, and calculated according to the Michaelis-Menten model, applying the Eadie-Hofstee linearization. Possible product inhibition of l-arabinose isomerase by d-tagatose was tested for initial concentrations of 0–500 mM.
3.8. Applications with Substrates
The native enzymes and the immobilized derivatives were used in three-step or one-step batch processes with the following substrates in 0.1 M phosphate buffer: d-glucose (100 g/L), d-galactose (100 g/L), lactose (46 g/L, equivalent to the concentration of lactose generally available in non-fermented fluid dairy products), and Mozzarella cheese whey. In the sequential three-step application, after lactose was hydrolyzed by β-galactosidase (step 1), the hydrolysis products were partially isomerized to d-tagatose and d-fructose by l-arabinose isomerase (step 2) and d-xylose isomerase (step 3). In the simultaneous processes, the three activities operated in a single bio-reactor. The activity of the immobilized enzyme derivatives was determined under different conditions of pH and temperature, and compared with the native enzymes.
In conclusion, β-galactosidase from B. circulans, l-arabinose isomerase from E. faecium, and d-xylose isomerase from S. rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L with high immobilization yields (around 100%) and high immobilized activity yields (around 80%–90%). Decrease in expressed activity after stabilization by a combined strategy (alkaline incubation for 24 h followed by blocking with glycine) was compensated for by the significant gain in thermal stability. This effect was most important in the case of the very sensitive l-arabinose isomerase, with half-lives at 50 °C of 308 and 379 h for Eupergit C 250 L and Eupergit C derivatives, respectively. These stabilized derivatives could work at moderately high temperatures, favoring tagatose production. The homotetrameric structure of d-xylose (d-glucose) isomerase and l-arabinose (d-galactose) isomerase could explain their higher stabilization levels with respect to monomeric β-galactosidase.
Sequential application of single-immobilizates of β-galactosidase, l-arabinose isomerase, and d-xylose isomerase to biotransform lactose at pH 7.0 achieve conversion percentages in terms of d-tagatose and d-fructose of 31% and 24%, respectively after 6 h operation at 50 °C. Soluble enzymes under a similar operation mode had lower productivity and conversion degrees than using the immobilized biocatalysts, showing better performance of immobilizates. However, the sequential use of the biocatalysts resulted in incomplete lactose hydrolysis because of product inhibition by d-galactose and d-glucose. On the other hand, a mono-reactor system with a combination of the three derivatives favored higher conversion percentages, especially for the lactolysis reaction, resulting in a strong increase in tagatose productivity (yield of 9.1 g/Lh) and a moderate increment in fructose productivity.
Tri-enzymatic co-immobilized derivatives could be more efficient biocatalysts to minimize or to avoid product inhibition during mono-reactor operation, but diverse conditions must be analyzed in detail with relation to coupling, stabilization, and operation mode. These studies are the aims of a forthcoming paper.